Abstract

Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl–transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.

Bacterial elongation factor P (EF-P) and its archaeal/eukaryotic homolog, initiation factor 5A (a/eIF5A), are universally conserved proteins with unknown cellular function. EF-P was reported to modulate cell viability, growth, virulence, motility, and sensitivity to low osmolarity, detergents, and antibiotics (1, 2). The EF-P binding site on the ribosome is located at the interface of the 30S and 50S ribosomal subunits between the binding sites for peptidyl-tRNA (P site) and the exiting tRNA (E site). The N-terminal domain of EF-P interacts with the acceptor stem of the P site–bound tRNA near the peptidyl transferase center (3). Both EF-P and eIF5A are posttranslationally modified (4–8). In Escherichia coli EF-P, Lys34 is modified by the action of three enzymes, YjeK, YjeA, and YcfM (5, 7–9). The proposed cellular function of EF-P/eIF5A is to optimize the ribosomes for more productive interactions with tRNA and release factors (3, 10–13). However, the magnitude of the reported effects appeared too small (less than twofold) for a universal function. We clarify the functional role of EF-P by investigating the role of the factor in the translation of various mRNA sequences.

The activity of EF-P in accelerating peptide bond formation was originally identified by its ability to increase the yield of formylmethionyl-puromycin (fMet-Pmn) synthesis (12, 13). Because reaction rates vary by ~1000-fold for different C-terminal amino acids in the peptidyl-tRNA (14), we examined whether EF-P may specifically accelerate the product formation with poorly reactive peptidyl-tRNAs, such as those containing C-terminal proline residues (Fig. 1A and fig. S1). The formation of the tripeptide fMetPro-Pmn was accelerated by almost 90-fold in the presence of EF-P, whereas in the other cases the reaction was stimulated by less than fivefold. To explore the effect of the amino acid following a proline residue, we tested Gly- and Pro-tRNAs that are known to be slow in peptide bond formation and can contribute to ribosome stalling (15–17). Whereas EF-P enhanced peptide-bond formation by less than twofold for most combinations, large effects were observed for the formation of fMetProGly (fMPG, eightfold) and fMetProPro (16-fold) (Fig. 1B). When longer strings of Pro and Gly residues, such as fMPPG, fMPPGF, or fMPPPF, were examined, practically no correct product was formed in the absence of EF-P, whereas the addition of EF-P rescued the rapid production of the respective peptide (Fig. 1C). The low yield of product formation in the absence of EF-P can be explained by the loss of peptidyl-tRNA from the stalled ribosomes (e.g., fMPP-tRNA; fig. S2A). These results suggested a specific effect of EF-P in accelerating the reaction with poorly reactive substrates that otherwise cause ribosome stalling.

EF-P promotes the synthesis of Pro- and Gly-containing peptides on the ribosome. (A) Rates (kobs) of peptide-bond formation between fMet-tRNAfMet or fMetX-tRNAX and Pmn, where X stands for different amino acids, as indicated, in the absence (white) or presence (black) of EF-P (3 μM). Error bars indicate standard deviations. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; E, Glu; F, Phe; G, Gly; K, Lys; M, Met; P, Pro; Q, Gln; R, Arg; V, Val; and W, Trp. (B) Rates of peptide-bond formation between fMet-tRNA (fM) or fMetPro-tRNA (fMP) in the P site and Gly-tRNAGly, Phe-tRNAPhe, or Pro-tRNAPro (G, F, and P, respectively) in the A site. (C) Formation of model oligopeptides in a reconstituted translation system in the absence (open) and presence (solid) of EF-P.

To verify whether proline-containing sequences as such induce ribosome stalling that can be rescued by EF-P, we engineered PG, PP, PPG, and PPP sequences into an N-terminal fragment of protein PrmC, which originally does not contain such sequences and is rapidly synthesized independent of the presence of EF-P (Fig. 2). Introducing a glycine residue after Pro20 resulted in the ribosome pausing, as shown by the accumulation of a peptide of about 20 amino acids; however, pausing was transient and not affected by EF-P. When a second proline residue preceding Pro20 was engineered, the ribosome paused at the ProPro sequence, and EF-P reduced the pause time from 20 to 10 s. When PPG or PPP sequences were introduced, the ribosomes were stalled, and essentially no full-length product was produced. The ribosomes stalled upon translation of the PPG sequence that contained a ProPro-ending peptidyl-tRNA in the P site and a Gly-tRNA in the A site (fig. S2B). Addition of EF-P rescued the synthesis of full-length product by preventing or alleviating ribosome stalling (Fig. 2 and fig. S2C).

The sequences PPG and PPP, as well as longer proline stretches, are found in a large number of cellular proteins, raising the question of whether they require EF-P for their synthesis. We tested the effect of EF-P on the synthesis of some of these proteins, such as TonB, YafD, Rz1, and AmiB (Fig. 3). In the absence of EF-P, translation was stalled at the consecutive prolines, and the addition of EF-P alleviated ribosome stalling, resulting in the rapid synthesis of the respective full-length peptide. When the Pro stretches were particularly long, as in AmiB or Rz1, very little full-length product was synthesized in the absence of EF-P, because translation was halted at the stalling site. In all cases, the addition of EF-P resulted in the efficient synthesis of full-length protein. Transient pauses that occurred at sequences other than PPP and PPG were not alleviated by EF-P (Fig. 3).

Posttranslational modification of EF-P appears crucial for the factor’s function (9, 18). Translation of the fMPPG sequence, which was abolished in the absence of EF-P, was fully restored with EF-P carrying the β-lysine modification at Lys34; further hydroxylation did not alter the activity, whereas the absence of modifications reduced the activity of the factor (Fig. 4). A similar effect was observed for the translation of the PPG sequence in PrmC (fig. S4). Analysis of the reaction kinetics indicated that the modification increased the affinity of EF-P binding to the ribosome 30-fold (Fig. 4). The maximum rate of fMPPG synthesis decreased ~fourfold with unmodified compared with modified EF-P, resulting in a >100-fold difference in kcat/KM(where kcat is the maximum reaction rate obtained by hyperbolic fittingand KM is the Michaelis constant). The low catalytic proficiency of unmodified EF-P may explain why deletions of the yjeA or yjeK genes that code for the modification enzymes lead to phenotypes that are similar to, or only somewhat milder than, the deletion of efp, the gene coding for EF-P (1, 2). On the other hand, even in the absence of active EF-P, small amounts of proteins with proline stretches are slowly formed (Fig. 3); this may be sufficient to support the viability of strains in which the efp, yieA, and yieK genes are deleted.

Our data indicate that EF-P is a translation factor that promotes the synthesis of proteins containing PPG, PPP, and longer polyproline stretches by preventing ribosome stalling during the formation of proline-proline or proline-glycine peptide bonds. Thus, EF-P (and likely eIF5A) can be considered a third universal translation elongation factor that acts on the ribosome, in addition to the two factors, EF-Tu and EF-G, that catalyze the mRNA decoding and tRNA translocation steps, respectively. In contrast to the latter two factors, which act in each elongation cycle irrespective of the amino acid incorporated, EF-P and eIF5A are responsible for the elongation of a subset of nascent proteins and are the only factors identified so far that augment the peptidyl transferase activity of the ribosome.

The pyrrolidine ring of proline restricts the number of accessible conformations for this residue and may impose structural constraints on the positioning of the amino acid in the peptidyl transferase center, resulting in slow peptide-bond formation and thereby ribosome stalling. EF-P facilitates peptide-bond formation by stabilizing peptidyl-tRNA on the ribosome and possibly by promoting optimal positioning of the substrates. The cellular fate of the nascent peptide on stalled ribosomes is likely to depend on its length: Shorter peptides may be lost because peptidyl-tRNA drop off the ribosome (16, 19), whereas ribosomes stalled after the synthesis of longer peptides might be rescued through SsrA-, ArfA-, or YaeJ-dependent pathways (20–23).

Of more than 4000 annotated E. coli proteins, about 270 contain motifs of three or more consecutive prolines or PPG motifs (table S1). Among those, proteins belonging to the basal transcription-translation machinery are underrepresented, whereas metabolic enzymes, transporters, and regulatory transcription factors are frequent. The observed pleiotropic effects of efp, yjeA, or yjeK deletions (1) can be rationalized by the impaired synthesis of proteins with proline stretches. For example, decreased synthesis of TonB, a protein from the EF-P interactome (24) that supplies energy for the function of TonB-dependent transporters, may result in a cumulative inhibition of transport processes (25). The severe effects of efp, yjeA, or yjeK deletions on cell motility may be explained by the failure to support the coordinated expression of flagellar proteins FlhC, FliF, and Flk, all containing PPP and PPG motifs, whereas the deficiency of EF-P mutants in using γ-glutamyl-glycine as a nitrogen source (1) may be caused by impaired synthesis of the corresponding catabolic enzyme, γ-glutamyl-transpeptidase, which contains a PPP motif. Lastly, EF-P may affect the translation of virulence proteins, such as protein EspF, which is a key player during the infection of eukaryotic hosts by enterohaemorrhagic and enteropathogenic strains of E. coli (26, 27). EspF contains several proline runs, including PPPP sequences, in its functionally important Src homology 3 binding domains (27). Given that the posttranslational modifications of eukaryotic eIF5A and bacterial EF-P are different, the occurrence of proline runs in many pathogenic proteins makes EF-P and its modification enzymes promising new targets for developing highly specific, potent antimicrobials.

Acknowledgments: We thank W. Wintermeyer for critically reading the manuscript; W. Holtkamp and J. Mittelstaet for the translation assay and M1 and M2 peptide markers; D. Görlich and C. Enke for the antibody against EF-P; and O. Geintzer, S. Kappler, T. Wiles, M. Zimmermann, T. Uhlendorf, A. Bursy, and U. Plessmann for expert technical assistance. The work was funded by the Max Planck Society and by grants from the Deutsche Forschungsgemeinschaft.